Process for the preparation of (r)- or (s)-aminocarnitine inner salt, the salts and derivatives thereof

ABSTRACT

A process for the production of (R)- or (S)-aminocarnitine starting respectively from (R)- or (S)-nitryloxycarnitine, through the formation and hydrogenation of the azidocarnitine intermediate with the same absolute configuration is described. (R)-aminocarnitine inner salt is obtained after purification, which is then converted into non-deliquescent salts. Also a process for the preparation of derivatives of (R)- and (S)-aminocarnitine, in particular acylayed or ureic derivatives, having known pharmacological properties, starting from (R)- and (S)-aminocarnitine salts, releasing the aminic function in situ.

This application is the U.S. national phase of international applicationPCT/IT02/00468, filed in English on 17 Jul. 2002, which designated theU.S. PCT/IT02/00468 claims priority to IT Application No. RM2001A000456filed 26 Jul. 2001. The entire contents of these applications areincorporated herein by reference.

The present invention relates to a process for the production of (R)-and (S)-aminocarnitine and salts and derivatives thereof. Thesecompounds or their derivatives, in particular those having an absolute(R) configuration exhibit interesting pharmacological properties;furthermore (R)- and (S)-aminocarnitine constitute useful chiralsynthons for the production of other enantiomerically pureintermediates.

BACKGROUND OF THE INVENTION

Jenkins D. and Griffith O. (P.N.A.S. U.S.A., 1986, 83(2), 290-4) havedescribed the antiketogenic and hypoglycaemic properties ofaminocarnitine. Other effects are described in Deana R. et al. Biol.Reprod., 1989, 41(5), 949-55, Jensen H et al. Biochim. Biophys. Acta(1990), 1044 (3), 390-3, Nagy I. et al. Pharmacol. Res. (2000), 41(1),9-17.

Aminocarnitine and its acetyl derivative were described in the U.S. Pat.No. 4,521,432, Takeda Chemical Industries, as a substance capable ofinhibiting the degradation of fatty acids, therefore useful as anantidiabetic agent. The substances described in this reference areobtained through the cultivation of strains of Emericella or Aspergillusand the complete synthesis of the aminocarnitine compound asconfirmation of the structure of the compound isolated from the strainsof micro organisms is also described.

In WO 85/04396, Cornell University, acylated derivatives ofaminocarnitine useful in the treatment of diabetes are described, thanksto their activity as inhibitors of carnitine acyltransferase. The acylderivatives are prepared by starting from aminocarnitine, thepreparation of which is provided through the deacetylation of thecorresponding acetylaminocarnitine or by starting from 4-bromocrotonate.

U.S. Pat. Nos. 4,767,781 and 4,948,534, related to U.S. Pat. No.4,521,432, describe further aminocarnitine derivatives, obtainable fromthe latter.

In the patent application WO 99/59957, ureic-structured derivativeshaving activities as reversible inhibitors of carnitinepalmitoyltransferase are prepared starting from (R)-aminocarnitine.

Numerous processes for the preparation of (R)- and (S)-aminocarnitineare known, which utilize various synthetic methodologies such as thedeacylation of the products obtainable through fermentation such asdescribed in U.S. Pat. No. 4,948,534, the use of chiral synthons such asaspartic acid, as described in WO 01/02341, in the name of theapplicant, the resolution of a racemic mixture, described in EP 0 402322. All these processes however have numerous defects such as lowproductivity, the high costs of some starting material, numeroussynthetic steps. An improved process which starts from mesyloxycarnitine methanesulphonate and its transformation into aminocarnitinevia the intermediate azidocarnitine has been recently proposed by theapplicant, as described in U.S. Pat. No. 5,532,409. In this process, themesyloxy carnitine methanesulphonate starting material is produced fromcarnitine in three steps with good yields (approx. 75%), but using acostly reagent such as methanesulphonic anhydride and producing, as acostly refluent, during the course of the synthesis of aminocarnitine,2-3 moles of aqueous methanesulphonic acid per mole of product. Theformation reaction of azidocarnitine is carried out under high dilutionand with consequent low productivity; furthermore the solvent used,DMSO, can present problems of partial instability in the alkalineconditions used in the recovery and recycling; the precipitating solventof the raw azide, ethyl ether, is industrially discouraged, in that itis dangerous. The 10% Pd/C catalyst used in the hydrogenation reactionhas a high precious metal content.

SUMMARY OF THE INVENTION

A process for the preparation of (R)- or (S)-aminocarnitine inner salt,its salts and their derivatives has now been found, starting fromrespectively (R)- or (S)-nitryloxycarnitine, through the formation andhydrogenation of the intermediate azidocarnitine of the same absoluteconfiguration.

Although a similar approach is used to that described in U.S. Pat. No.5,532,409 and obtaining a final product with a comparable yield, if oneconsiders the total yield starting from carnitine, the present processdoes not present the drawback discussed above and, as will be apparentfor those skilled in the art, offers numerous advantages. Amongst theseare cited the low costs, the improved productivity, the possibility ofnot having to isolate the azidocarnitine, the hydrogenation carried outwith a catalyst having a lower precious metal content, the use in theformation of azidocarnitine of an easily recoverable and recyclablesolvent, and the use of water for the hydrogenation reaction. Otheradvantages will be apparent in the following examples. Theaminocarnitine thus obtained can be further purified, if desired and thepurification of the aminocarnitine is carried out through the use ofnormal techniques known to those skilled in the art, amongst which arepreferred ion exchange resins or electrodialysis. This way, in additionto the inorganic salts, in particular sodium and potassium nitrates, thereaction by-products such as carnitine and crotonoyl betaine areremoved. Potassium nitrate can be largely removed through filtrationbeing poorly soluble in water and in the organic solvents used in theazidation reaction. The present process also offers the possibility toeasily recover, using an industrially acceptable method, theaminocarnitine in the form of a non-deliquescent salt, such as forexample sulphate. The use of such a salt offers the advantage, when theaminocarnitine is used as an intermediate for the preparation ofpharmacologically active acylated or ureic derivatives, to be able tofree the product in situ from the corresponding salts by alkalitreatment, avoiding every manipulation of the deliquescent inner salt.

DETAILED DESCRIPTION OF THE INVENTION

The process according to the present invention comprises the followingsteps:

-   -   a) transformation of the (R)- or (S)-nitryloxycarnitine        respectively in (R)- or (S)-azidocarnitine;    -   b) hydrogenation of the (R)- or (S)-azidocarnitine obtained in        step a) to give respectively (R)- or (S)-aminocarnitine and, if        desired    -   c) salification of the (R)- or (S)-aminocarnitine and, if        desired    -   d) transformation of the (R)- or (S)-aminocarnitine into one of        its derivatives.

The (R)- and (S)-nitryloxycarnitine can be prepared as described in thepatent application WO 01/10819, in the name of the applicant. Thesecompounds are available in the form of inner salts, and as the salts oforganic or inorganic acids, amongst which nitrate is preferred.

The reaction of step a), is carried out in an appropriate solventcompatible with the reagents and the end product. Among the suitablesolvents, polar aprotic solvents, such as for exampleN-methyl-pyrrolidone, dimethylacetamide and dimethylformamide arepreferred.

In a first preferred embodiment, (R)- or (S)-nitryloxycarnitine,preferably as nitrate, is dissolved in the solvent medium, for exampleN-methyl-pyrrolidone, and later treated, in the presence of a base, withan azotide acid salt, such as for example lithium or sodium azide. Apreferred base is sodium or potassium phthalimide.

The hydrogenation of step b) is carried out according to process knownto those skilled in the field, however, hydrogenation carried out usingPd/C as catalyst is preferred, for example at 3%. Preferably, thephthalimide is removed prior to the hydrogenation of step b), forexample by precipitation and filtration.

If desired, the aminocarnitine inner salt (i.s.) is converted into oneof its non-deliquescent and stable salts. In U.S. Pat. No. 4,948,534hydrochloride, sulphate, nitrate, oxalate, acetate, succinate, fumarate,citrate as aminocarnitine salts are cited. The hydrochloride, describedin example XX, is obtained by precipitation using a mixture of solvents,such as methanol and ethyl ether, and is purified by subsequentcrystallization from methanol. The use of a methanol and ethyl ethermixture is not however particularly desirable from an industrial pointof view, therefore, the present invention presents in one of itspreferred embodiments, a way to obtain stable and non-deliquescentaminocarnitine salts using a more industrially suitable solvent. By nondeliquescent or non-hygroscopic salt it is intended a salt which,without the need to resort to special process or the use of particularapparatus, does not absorb a quantity of water that would compromise theindustrial manageability for the normal manufacture and/or its storagein normal industrial conditions of the starting materials or thecompositions containing them. Among salts having these properties, theinorganic acid salts, and in particular sulphate are particularlypreferred, both because the corresponding acid is very economical, andbecause said salt is obtained with almost quantitative yield at a highgrade of chemical purity by precipitation of a single, industriallyadvantageous, solvent such as methanol, and because it is very simple,as those skilled in the field can easily see from the aminocarnitinei.s. regeneration process, given as an example below.

In another aspect of the present invention, (R)- or (S)-aminocarnitine,obtained according to the process of the present invention, and in theform of one of its salts, is further transformed into its derivatives,in particular acylated and ureic derivatives, as described for examplein WO 99/59957. This transformation, which process is a further objectof the present invention, comprises:

-   -   a) the release of the aminic group of the corresponding salt,        for example of the corresponding sulphate, by treatment with        alkaline metal or alkaline earth metal hydroxides or carbonates,        in situ, in a solvent suitable for the subsequent        functionalisation;    -   b) optional removal, for example by filtration, of the insoluble        products thus obtained, such as the alkaline metal or alkaline        earth metal sulphates;    -   c) the functionalisation of the aminic function by the addition,        still in situ, of the appropriate reagent.

This process is particularly advantageous if the functionalisationreagent, such as for example an isocyanate or a chloride acid, isdegraded partially or totally in the presence of water.

The following examples further illustrate the present invention.

EXAMPLE 1

Preparation of (R)-aminocarnitine Sulphate

(R)-nitryloxycarnitine nitrate (50 g, 186 mmol), prepared according topatent application WO 01/10819, is dissolved in N-methyl-pyrrolidone(NMP) (1000 ml; 1026 g) and to potassium phthalimide (41 g, 222 mmol)and sodium azide (12.1 g, 186 mmol) the solution are added in sequence.

After keeping the reaction mixture under stirring for 18 hours at roomtemperature, the solvent is distilled under reduced pressure (50-60° C.at 3-4 mmHg).

At this point, H₂O (88 ml) is added to the residue to precipitate thephthalimide which is filtered and washed.

Pd/C 3% (3.5 g) is added to the filtrate (435 g) and subjected tohydrogenation in an atmosphere of 100 p.s.i. of hydrogen pressure for8.5 hours at 25-26° C.

On termination of hydrogenation, 2 g of active C are added to thereaction mixture, and, after 15 minutes of stirring, filtered throughcelite to obtain an aqueous solution comprising the washes of approx.690 g aminocarnitine i.s. titre of 1.6% (approx. 11 g; 69 mmol, yield37%). The solution is first eluted on IRA 410 (OH—) (1000 ml) and thenon IRC 50 (COOH) (250 ml).

The washed eluates are recovered in sequence as (approx. 5 1, containingcarnitine and crotonoyl betaine as the main impurities), and anammoniacal eluate containing the product (approx. 1000 ml).

After having concentrated the latter to approx. 112 g (theaminocarnitine i.s. HPLC titre was 8.8%; 62 mmol, yield 33%) the eluateis acidified to pH 2 with 96% H₂SO₄ (6.2 g; 62 mmol).

At this point the concentration continues by azeotroping the water withisobutyl alcohol, finally adding methanol, to leave the(R)-aminocarnitine sulphate to precipitate in the warm (approx. 50° C.).After further concentration of the methanol to a mixture weight ofapprox. 105 g, this is cooled to room temperature and filteredrecovering 15.4 g dry weight (yield 32%) after desiccation.

M.p. (DSC): 217.5-218° C. dec.; [α]²⁵D:+6.37, (c=1%, H₂O), e.e. 97.8%

EXAMPLE 2

Preparation of (R)-aminocarnitine Sulphate

Operating as in example 1 but using 37.5 g of sodium phthalimide insteadof 41 g of potassium phthalimide and 1000 ml of DMF instead of NMP,after the hydrogenation reaction, an aqueous solution containingaminocarnitine i.s. with a yield of 30% is obtained. One then proceedsas in example 1 to obtain (R)-aminocarnitine sulphate.

EXAMPLE 3

Preparation of (R)-aminocarnitine Sulphate

Operating as in example 1 but precipitating the azide intermediate bytreatment with acetone or with butyl acetate, successively removing theorganic solvent residue under reduced pressure and dissolving theaqueous azide thus obtained, after the hydrogenation reaction, anaqueous solution containing aminocarnitine i.s. with a yield of 33% isobtained. One then proceeds as in example 1 to obtain (R)-aminocarnitinesulphate.

EXAMPLE 4

Preparation of (R)-N′-tetradecylcarbamoylaminocarnitine

To a solution of 1 g of (R)-aminocarnitine sulphate in 6 ml of water,KOH (0.33 g) is added. The mixture is concentrated under vacuum, takenup in methanol and concentrated again; the operation is repeated untilsubstantial anhydrification. 10 ml of methanol are then added and theinorganic salts filtered; after partial concentration to a volume ofapprox. 4 ml, the solution is cooled to 5° C. and tetradecylisocyanate(1 ml) added. After standing overnight at room temperature thein-soluble fraction is filtered, concentrated to dryness in methanol,and the residue is taken up in acetone (10 ml) and kept under stirringfor 2 hours. After filtration and drying approx. 1.3 g of(R)-N′-tetradecyl-carbamoyl aminocarnitine are obtained.

EXAMPLE 5

Preparation of (R)-N-acetyl-aminocarnitine

A mixture of aminocarnitine sulphate (2 g), sodium carbonate (1.3 g) in40 ml of NMP is kept stirring for 12 hours at room temperature, then 3.7g of acetic anhydride added and kept under stirring for a further 24hours to give (R)-N-acetylaminocarnitine.

1. A process for the preparation of (R)- or (S)-aminocarnitine innersalt, the salts and a derivatives thereof, said derivative beingselected from the group consisting of —NHR, —NHCSR, —NHCOOR, —NHCSOR,—NHCONHR, —NHCSNHR, -NHSOR, —NHSONHR, —NHSO₂R, —NHSO₂NHR, wherein R is aC₁-C₂₀ saturated or unsaturated, straight or branched alkyl group,optionally substituted with a A₁ group, wherein A₁ is selected from thegroup consisting of halogen atom, C₆-C₁₄ aryl, heteroaryl, aryloxy,heteroaryloxy, groups, being optionally substituted with one or moreC₁-C₂₀ saturated or unsaturated, straight or branched alkyl or alkoxygroup, and/or halogen atom, said process comprising: a) treating (R)- or(S)-nitryloxycarnitine with sodium or potassium phthalimide, then withlithium or sodium azide to give respectively (R)- or (S)-azidocarnitine;b) hydrogenating the (R)- or (S)-azidocarnitine obtained in step a) togive respectively (R)- or (S)-aminocarnitine and, if desired c)salifying the (R)- or (S)-aminocarnitine and, if desired d) transformingthe (R)- or (S)-aminocarnitine into one of said derivatives.
 2. Theprocess according to claim 1, wherein the aminocarnitine obtained isfurther purified.
 3. The process according to claim 2, wherein saidpurification is by treatment with ion exchange resins or throughelectrodialysis.
 4. The process according to claim 1, wherein theaminocarnitine is later converted into a non-deliquescent salt.
 5. Theprocess according to claim 4, in which said salt is sulphate.
 6. Theprocess according to claim 1, wherein a polar aprotic solvent medium isused in step a).
 7. The process according to claim 6, wherein saidsolvent is N-methyl-pyrrolidone, dimethylacetamide or dimethylformamide.8. A process for the preparation of derivatives of (R)- or(S)-aminocarnitine, which comprises: a) transforming the (R)- or(S)-nitryloxycarnitine respectively in (R)- or (S)-azidocarnitine; b)hydrogenating the (R)- or (S)-azidocarnitine obtained in step a) to giverespectively (R)- or (S)-aminocarnitine, c) transforming of the (R)- or(S)-aminocamitine of step b) into a non-deliquescent, non-hygroscopicsalt thereof; d) releasing of the aminic function of the correspondingsalt, by treating with alkaline metal or alkaline earth metal hydroxidesor carbonates, in situ, in a solvent suitable for the subsequentfunctionalization; e) optionally removing the insoluble products thusobtained; and f) in-situ functionalizing the released aminic function.9. The process according to claim 8, wherein said derivatives areacylated or ureic.
 10. The process according to claim 9, wherein saidderivatives are (R)- or (S)-N-acetylaminocarnitine or (R)- or(S)-N-tetradecyl carbamoyl aminocarnitine.
 11. The process according toclaim 9, wherein the salt of (R)- or (S)-aminocarnitine is treated witha base, then with an appropriate isocyanate to give the correspondingureic derivative.
 12. The process according to claim 9, wherein the (R)-or (S)-aminocarnitine salt is treated with a base, then with anappropriate acylating agent to give the corresponding acylic derivative.13. The process according to claim 8, wherein the salt of (R)- or(S)-aminocarnitine is sulphate.
 14. The process according to claim 8,wherein, in step c), said salt is sulphate.
 15. A process for thepreparation of (R)- or (S)-aminocarnitine inner salt, the salts and aderivative thereof, said derivative being selected from the groupconsisting of —NHR, —NHCSR, —NHCOOR, —NHCSOR, —NHCONHR, —NHCSNHR,—NHSOR, —NHSONHR, —NHSO₂R, —NHSO₂NHR, wherein R is a C₁-C₂₀ saturated orunsaturated, straight or branched alkyl group, optionally substitutedwith a A₁ group, wherein A₁ is selected from the group consisting ofhalogen atom, C₆-C₁₄ aryl, heteroaryl, aryloxy, heteroaryloxy, groups,being optionally substituted with one or more C₁-C₂₀ saturated orunsaturated, straight or branched alkyl or alkoxy group,and/or halogenatom, said process comprising: a) transforming the (R)- or(S)-nitryloxycarnitine respectively in (R)- or (S)-azidocarnitine; b)hydrogenating the (R)- or (S)-azidocarnitine obtained in step a) to giverespectively (R)- or (S)-aminocarnitine and, if desired c) transformingthe (R)- or (S)-aminocamitine of step b) into a non-deliquescent,non-hygroscopic salt thereof; and, if desired d) transforming the (R)-or (S)-aminocarnitine into one of said derivatives.
 16. The processaccording to claim 15, wherein the aminocarnitine obtained is furtherpurified.
 17. The process according to claim 16, wherein saidpurification is by treatment with ion exchange resins or throughelectrodialysis.
 18. The process according to claim 15, wherein, in stepb), said salt is sulphate.
 19. The process according to claim 15,wherein a polar aprotic solvent medium is used in step a).
 20. Theprocess according to claim 19, wherein said solvent isN-methyl-pyrrolidone, dimethylacetamide or dimethylformamide.
 21. Theprocess according to claim 19, wherein in step a) (R)- or(S)-nitryloxycarnitine is treated with sodium or potassium phthalimide,then with lithium or sodium azide.